Insulin resistance represents a loss or reduction in its normal functionality on target tissues and hence affects our cognitive and memory functions, ultimately leading to the onset of Alzheimer disease (AD) (Cholerton et al, 2011). Insulin resistance has been linked to several previously identified risk factors that accelerate the cognitive dysfunction and ageing process, including diabetes, obesity, hypertension, hyperlipidemia, and metabolic syndrome (Spielman et al, 2014). Particularly, brains exhibit defective insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) show alteration or aberrant activation of insulin signaling in progression of AD, the most common cause of dementia (Williamson et al, 2012). These findings suggest that neuronal insulin signaling becomes dysfunction in the AD brains similar to the dementia symptoms of Type 2 diabetes. The pathogenesis of AD is initially triggered by the presence of extracellular amyloid-β (Aβ) peptides, which impair mitochondrial membrane potential (MMP) and contribute to an increase in the accumulation of intracellular reactive oxygen species (ROS), ultimately leading to neuronal cell death (Butterfield D A, 2002; Li et al, 2015). It has been well established that Aβ deposition may play a pathogenic role in age-associated AD pathogenesis (Lesne et al, 2013). In addition, our previous studies have indicated that Aβ induces p-Ser307 IRS-1 expression and inhibits IRS-1 tyrosine phosphorylation and its downstream target protein kinase B (PKB, also called Akt) (Kornelius et al, 2015). Subsequently, Aβ further suppresses Ser9 phosphorylation of glycogen synthase kinase 3β (GSK3β), which is one of the enzymes responsible for causing tau hyperphosphorylation and neurotoxicity (Hernandez et al, 2013). These findings all indicate that insulin signaling plays a key regulatory role in Aβ-induced neurotoxicity and neuronal cell death in AD patients.
Cell survival is maintained by external factors such as growth factors, the lack of which often causes apoptosis. The Akt signaling pathway has been reported as a major downstream effector of growth factor-mediated cell survival mechanisms that inhibit apoptosis (Bhat and Thirumangalakudi, 2013). To this, Akt functions to promote cell survival by inactivating certain pro-apoptotic mediators such as Bid, a pro-apoptotic member of the Bcl-2 family involved in the induction of death receptor-mediated apoptosis (Majewski et al, 2004). Also, Akt signaling can reduce oxidative stress via activating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) antioxidant pathway (Surh et al, 2008), subsequently leading to prevention of Aβ-induced neurotoxicity (Kwon et al, 2015). As a result, both of these reported Akt-mediated protective mechanisms against cell apoptosis and oxidative stress may be useful for preventing neurodegeneration and mitochondrial dysfunction in human brains. Interestingly, in human embryonic stem cells (hESC), microRNA miR-302 has been found to mediate Akt activation through downregulating phosphatase and tensin homolog (PTEN) in order to maintain the pluripotency of hESCs (Alva et al, 2011). Moreover, Akt signaling also regulates the pluripotency-associated gene Nanog to maintain stem cell self-renewal and anti-ageing (Kuijk, 2010; Han et al, 2012). Taken together, based on all the above findings, we have proposed that miR-302 may be able to stimulate the activation of the Akt signaling pathway in neurons, so as to prevent Aβ-induced neurotoxicity in AD patients. Yet, neurons as one type of somatic cells normally do not express miR-302.
MicroRNA (miRNA) miR-302 is the most abundant non-coding RNA species specifically found in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Our previous studies have shown that ectopic expression of miR-302 in mammalian somatic cells is able to reprogram the somatic cells to hESC-like iPSCs (as demonstrated in Lin et al., 2008, 2010 and 2011; EP 2198025; U.S. Ser. No. 12/149,725; U.S. Ser. No. 12/318,806; U.S. Ser. No. 12/792,413). Moreover, we have also observed that introduction of miR-302 into mammalian cells can further stimulate the expression of many other miRNA species, such as miR-92, miR-93, miR-367, miR-369, miR-371373, miR-374, miR-517, and the whole miR-520 familial members (Lin et al., 2008, 2010 and 2011; EP 2198025; U.S. Ser. No. 12/149,725; U.S. Ser. No. 12/318,806; U.S. Ser. No. 12/792,413). Further analyses using the online “TARGETSCAN” and “PICTAR-VERT” programs, published in the Sanger Institute miRBase website (http://www.mirbase.org/), revealed that miR-302 shares over 400 target genes with these stimulated miRNAs, suggesting that they may play a similar or partially functional role like miR-302. Based on ours and many other previous reports, these shared target genes include, but not limited, members of RAB/RAS-related oncogenes, ECT-related oncogenes, pleiomorphic adenoma genes, E2F transcription factors, cyclin D binding Myb-like transcription factors, HMG-box transcription factors, Sp3 transcription factors, transcription factor CP2-like proteins, NFkB activating protein genes, cyclin-dependent kinases (CDKs), MAPK/JNK-related kinases, SNF-related kinases, myosin light chain kinases, TNF-alpha-induce protein genes, DAZ-associated protein genes, LIM-associated homeobox genes, DEAD/H box protein genes, forkhead box protein genes, BMP regulators, Rho/Rac guanine nucleotide exchange factors, IGF receptors (IGFR), endothelin receptors, left-right determination factors (Lefty), cyclins, p53 inducible nuclear protein genes, RB-like 1, RB binding protein genes, Max-binding protein genes, c-MIR cellular modulator of immune recognition, Bcl2-like apoptosis facilitator, protocadherins, TGFβ receptors, integrin β4/β8, inhibin, ankyrins, SENP1, NUFIP2, FGF9/19, SMAD2, CXCR4, EIF2C, PCAF, MECP2, histone acetyltransferase MYST3, nuclear RNP H3, and many nuclear receptors and factors. Notably, the majority of these target genes are highly involved in embryonic development and cancer tumorigenecity. Hence, it is conceivable that miR-302 can stimulate these downstream homologous miRNAs, such as miR-92, miR-93, miR-367, miR-371373, miR-374, and miR-520s, to enhance and/or maintain its functionality.
Particularly, we noted that miR-302, miR-9293, miR-367, miR-371374, and miR-520s are all hESC-specific miRNAs that are abundantly expressed in hESCs and iPSCs (Lin et al, 2008; EP 2198025; U.S. Ser. No. 12/149,725), all of which are also useful for designing and developing novel regenerative medicine. To achieve this goal, stem cells such as hESCs and iPSCs can be used as a treasure box as well as a tool for us to screen, search, extract, and produce novel effective drug-like ingredients that are useful for designing and developing many pharmaceutical and therapeutic applications, including but not limited, for stimulating tissue/organ regeneration, for repairing and/or rejuvenating damaged/aged cells/tissues, for treating ageing-associated degenerative diseases (i.e. Alzheimer's diseases, Parkinson's diseases, osteoporosis, diabetes and cancers), and for preventing tumor and/or cancer formation/progression/metastasis. As a result, it is conceivable that we can use these hESC-specific miRNAs as candidate drugs for developing novel therapies and treating human diseases in vivo. To fulfill this goal, we need a method for producing a significantly large amount of hairpin-like miRNAs and their precursors (pre-miRNAs) using modern DNA recombination and amplification technologies with bacterial cells; yet, it has been widely known that hairpin-like DNA/RNA structures resemble signals of intrinsic transcription termination mechanisms in prokaryotes (McDowell et al., Science 1994) and hence make it impossible for prokaryotic cells to transcribe hairpin-like RNAs, such as small hairpin RNAs (shRNA), microRNAs (miRNA) and the related precursors (i.e. pre-miRNA). To this problem, neither the first finder of miR-302—Houbaviy et al. (Developmental Cell (2003) 5, 351-358) nor the next follower Kim et al. (WO 2005/056797) could provide any solution for it.
Furthermore, as learning from current textbooks, a person of ordinary skill in the art must know that prokaryotic and eukaryotic transcription machineries are different and thus are not compatible to each other in many aspects. For example, based on most current understandings, eukaryotic RNA polymerases do not directly bind to gene promoter sequences and hence require additional accessory proteins to help it to initiate RNA transcription, whereas prokaryotic RNA polymerases can form a holoenzyme that binds directly to gene promoters, so as to initiate RNA transcription. However, because the holoenzyme can not process through a DNA sequence with a high degree of secondary structures, such as a hairpin DNA, the prokaryotic promoters naturally do not contain any hairpin-like structure, which otherwise resembles a transcription termination code in prokaryotes (McDowell et al, 1994). In addition, it is a common sense for a person of ordinary skill in the art to understand that eukaryotic messenger RNA (mRNA) is transcribed in the nucleus by type II RNA polymerases (pol-2) and then processed and exported to the cytoplasm for protein synthesis, whereas prokaryotic RNA transcription and protein translation take place simultaneously off the same piece of DNA in the same place (cytoplasm) because prokaryotic cells such as bacteria and archaea do not possess any nucleus-like structure. Due to these differences, it makes prokaryotes difficult or even impossible to produce eukaryotic RNAs and the related peptides/proteins using eukaryotic RNA promoters, which tend to contain DNA motifs with specific secondary structures in the 5′-untranslational regions (5′-UTR).
Prior arts attempt at producing mammalian gene products in bacterial cells, such as U.S. Pat. No. 7,959,926 to Buechler and U.S. Pat. No. 7,968,311 to Mehta, used bacterial or bacteriophage promoters. Since prokaryotes do not contain any splicing machinery such as spliceosome to process introns, the intron-less complementary DNA (cDNA) of a desired gene was made and cloned into a plasmid vector driven by a bacterial or bacteriophage promoter. Then, the vector so obtained was introduced into a competent strain of bacterial cells, such as Escherichia coli (E. coli), for expressing the gene transcripts (i.e. mRNAs) and subsequently translating the mRNAs into proteins. Nevertheless, the bacterial and bacteriophage promoters, such as Tac, Lac, Tc, T1, T3, T7, and SP6 RNA promoters, are not pol-2 promoters and their transcription activities tend to be an error-prone process, which causes mutations and can not express hairpin-like miRNAs or shRNAs as reported by McDowell et al (Science 1994). In addition, Mehta further taught that glycerol/glycerin might be used to increase the efficiency of bacterial transformation; yet, no teaching was related to enhancement of RNA transcription, in particular pol-2 promoter-driven hairpin-like RNA transcription. Due to lack of compatibility between eukaryotic and prokaryotic transcription systems, these prior arts were still limited by the use of prokaryotic RNA promoters for expressing gene cDNA in prokaryotes and none of them were useful for expressing hairpin-like RNAs, such as miRNAs and shRNAs.
Using a novel hairpin-RNA transcription mechanism newly found in prokaryotes (Lin et al, U.S. patent application Ser. No. 13/572,263, Ser. No. 14/502,608, and Ser. No. 14/527,439), we now can overcome the prokaryotic transcription termination mechanisms and thus induce over-expression of hairpin-like microRNA precursors (pre-miRNA) and shRNAs in prokaryotic cells, particularly useful for expressing human miR-302 familial microRNAs (miR-302a, b, c, d, e, and f) and their precursors (pre-miR-302). By adding certain transcriptional inducer chemicals into bacterial culture medium, we are able to transform prokaryotes to adopt eukaryotic pol-2 and/or viral pol-2-like promoters for transcribing our desired hairpin RNAs and the related miRNAs/shRNAs thereof. The advantages of this production method are: first, cost-effective production due to the fast and cheap growth of single-cell prokaryotes such as bacterial cells; second, easy handling because of no need for culturing dedicate hybridomas or mammalian cells; third, high product quality in view of the improved reading fidelity of pol-2 promoter-driven transcription; fourth, industrial level bulk production for desired hairpin RNAs and their related miRNAs/shRNAs as well as the introduced vectors all at once in prokaryotes; and last, multiple task capacity in that the desired RNAs and other desired peptides/proteins can be produced together but separately isolated and purified from the resulting bacterial extracts and/or lysates for further applications. Therefore, taken together, a composition and method for producing hairpin RNAs and/or their related miRNAs/shRNAs using eukaryotic RNA promoter-driven transcription in prokaryotes is highly desirable for the need of mass production of hairpin-like RNA drugs.